Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib.

Mature circulating endothelial cell (CEC) as well as endothelial progenitor populations may reflect the activity of anti-angiogenic agents on tumor neovasculature or even constitute a target for anti-angiogenic therapy. We investigated the behavior of CECs in parallel with hematopoietic progenitor cells (HPCs) in the blood of renal cell cancer patients during sunitinib treatment. We analyzed the kinetics of a specific population of small VEGFR2-expressing CECs (CD45(neg)/CD34(bright)), HPCs (CD45(dim)/CD34(bright)), and monocytes in the blood of 24 renal cell cancer (RCC) patients receiving 50 mg/day of the multitargeted VEGF inhibitor sunitinib, on a 4-week-on/2-week-off schedule. Blood was taken before treatment (C1D1), on C1D14, C1D28, and on C2D1 before the start of cycle 2. Also plasma VEGF and erythropoietin (EPO) were determined. Remarkably, while CD34(bright) HPCs and monocytes decreased during treatment, CD34(bright) CECs increased from 69 cells/ml (C1D1) to 180 cells/ml (C1D14; P = 0.001) and remained high on C1D28. All cell populations recovered to near pre-treatment levels on C2D1. Plasma VEGF and EPO levels were increased on C1D14 and partly normalized to pre-treatment levels on C2D1. In conclusion, opposite kinetics of two circulating CD34(bright) cell populations, HPCs and small CECs, were observed in sunitinib-treated RCC patients. The increase in CECs is likely caused by sunitinib targeting of immature tumor vessels.

Sunitinib-induced myeloid lineage redistribution in renal cell cancer patients: CD1c+ dendritic cell frequency predicts progression-free survival.

PURPOSE: A disturbed myeloid lineage development with abnormally abundant neutrophils and impaired dendritic cell (DC) differentiation may contribute to tumor immune escape. We investigated the effect of sunitinib, a tyrosine kinase inhibitor of fms-like tyrosine kinase-3, KIT, and vascular endothelial growth factor receptors, on myeloid differentiation in renal cell cancer (RCC) patients. EXPERIMENTAL DESIGN: Twenty-six advanced RCC patients were treated with sunitinib in a 4-week on/2-week off schedule. Enumeration and extensive phenotyping of myeloid subsets in the blood was done at baseline and at weeks 4 and 6 of the first treatment cycle. Baseline patient data were compared with sex- and age-matched healthy donor data. RESULTS: Baseline frequencies of DC subsets were lower in RCC patients than in healthy donors. After 4 weeks of sunitinib treatment, a generalized decrease in myeloid frequencies was observed. Whereas neutrophils and monocytes, which were both abnormally high at baseline, remained low during the 2-week off period, DC rates recovered, resulting in a normalized myeloid lineage distribution. Subsequent to sunitinib treatment, an increase to high levels of myeloid DC (MDC) subset frequencies relative to other myeloid subsets, was specifically observed in patients experiencing tumor regression. Moreover, high CD1c/BDCA-1(+) MDC frequencies were predictive for tumor regression and improved progression-free survival. CONCLUSION: The sunitinib-induced myeloid lineage redistribution observed in advanced RCC patients is consistent with an improved immune status. Immunologic recovery may contribute to clinical efficacy as suggested by the finding of highly increased MDC frequencies relative to other myeloid subsets in patients with tumor regression.

Celecoxib enhances doxorubicin-induced cytotoxicity in MDA-MB231 cells by NF-kappaB-mediated increase of intracellular doxorubicin accumulation.

Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclo-oxygenase (COX) inhibitors are anti-inflammatory agents that have also shown to be useful in anticancer therapy. In the present study, we show that the specific COX-2 inhibitor celecoxib enhances the inhibitory effect of doxorubicin (dox) on human MDA-MB231 breast tumour growth in vivo and in vitro. We also found that celecoxib increased the intracellular accumulation and retention of dox in vitro. Since the NSAID indomethacin and the specific COX-2 inhibitor NS398 did not affect the in vitro actions of dox, these effects are likely to be mediated via a COX-independent mechanism. It has been suggested that some COX-inhibitors can enhance the actions of cytostatics by overcoming multidrug resistance through the inhibition of ABC-transporter proteins. However, we found that the three main ATP-binding cassette (ABC)-transporter proteins, implicated in dox transport, were inactive in MDA-MB231 cells. Therefore, the finding that the P-glycoprotein (P-gp) blocker PSC833 also increased cellular accumulation of dox was unexpected. In order to unravel the molecular mechanisms involved in dox accumulation, we examined the involvement of NF-kappaB, as this transcription factor has been implicated in celecoxib action as well as in chemoresistance. We found that celecoxib and PSC833, but not indomethacin or NS398, almost completely inhibited basal- and dox induced NF-kappaB gene-reporter activity and p65 subunit nuclear translocation. Furthermore, the NF-kappaB inhibitor PDTC mimicked the actions of celecoxib and PSC833 on cell growth and on intracellular accumulation of dox, suggesting that NF-kappaB is functionally involved in the actions of these compounds. In conclusion, we show that structurally different compounds, among which are celecoxib and PSC833, increase the intracellular accumulation of dox and enhance dox induced cytotoxicity in MDA-MB231 breast cancer cells most likely via the modulation of NF-kappaB activity.

VEGFR2 expressing circulating (progenitor) cell populations in volunteers and cancer patients.

Circulating cells of several lineages are thought to participate in angiogenesis and tumor growth. Experimental studies in tumor-bearing mice have pointed to the potential importance of VEGF-responding circulating (endothelial) progenitor cells in tumor growth. We have studied circulating CD31- and/or CD34-positive cell populations with a low to moderate VEGFR2 expression in human volunteers and cancer patients. We recognized four cell populations, which were further characterized by their content of major hematopoietic progenitor, monocytic, endothelial and platelet markers. After establishing the test-retest stability of the measurements in nine patients, we determined the frequencies of the various cell populations in a group of 20 volunteers and 14 cancer patients. Two populations were markedly increased in cancer patients. Small CD45(neg)/CD34(bright)/VEGFR2+ cells amounted to 12 and 64 cells/ml (P < 0.0001), respectively, and 246/ml and 578/ml VEGFR2+/CD45(bright) (/CD14+) monocytic cells were present in controls and cancer patients, respectively (P = 0.017). A third population of CD45(dim)/CD34(bright)/VEGFR2(low) cells amounted to 25 and 30 cells/ml (P = 0.38). Unexpectedly, a population of mainly anucleated CD45(low)/CD31(bright)/CD41(bright) cells was present in numbers of 9,076 and 16,697/ml (P = 0.04) in volunteers and cancer patients, which contained a VEGFR2(low) (compared to IgG isotype control) expressing population amounting to 1,142 and 1,642 cells/ml (P = 0.12). This fourth population probably reflects large platelets. The role of the herein identified VEGFR2+ circulating cell populations deserve further investigation in cancer patients treated with VEGF(R)-targeted therapies. Quantification of such cell populations in the blood of tumor patients may be valuable to monitor the efficacy of anti-angiogenic treatment.

Argpyrimidine-modified Heat shock protein 27 in human non-small cell lung cancer: a possible mechanism for evasion of apoptosis.

Tumors generally display a high glycolytic rate. One consequence of increased glycolysis is the non-enzymatic glycation of proteins leading to the formation of advanced glycation end-products (AGEs). Therefore, we studied the presence of AGEs in non-small cell lung cancer and consequences thereof. We show the presence of two AGEs, i.e. the major AGE N(epsilon)-(carboxymethyl)lysine (CML) and the methylglyoxal-arginine adduct argpyrimidine, in human non-small cell lung cancer tissues by immunohistochemistry. We found in squamous cell carcinoma and adenocarcinoma tissues a strong CML positivity in both tumour cells and tumour-surrounding stroma. In contrast, argpyrimidine positivity was predominantly found in tumor cells and was strong in squamous cell carcinomas, but only weak in adenocarcinomas (2.6+/-0.5 vs. 1.2+/-0.4, respectively; P<0.005). In accordance, argpyrimidine was found in the human lung squamous carcinoma cell line SW1573, while it was almost absent in the adenocarcinoma cell line H460. Heat shock protein 27 (Hsp27) was identified as a major argpyrimidine-modified protein. In agreement with a previously described anti-apoptotic activity of argpyrimidine-modified Hsp27, the percentage of active caspase-3 positive tumor cells in squamous cell carcinomas was significantly lower when compared to adenocarcinomas. In addition, incubation with cisplatin induced almost no caspase-3 activation in SW1573 cells while a strong activation was seen in H460 cells; which was significantly reduced by incubation with an inhibitor of glyoxalase I, the enzyme that catalyzes the conversion of methylglyoxal. These findings suggest that a high level of argpyrimidine-modified Hsp27 is a mechanism of cancer cells for evasion of apoptosis.

Dose-finding study of the multitargeted tyrosine kinase inhibitor SU6668 in patients with advanced malignancies.

PURPOSE: SU6668 is a tyrosine kinase inhibitor which targets platelet-derived growth factor receptor-beta, fibroblast growth factor receptor-1, vascular endothelial growth factor receptor-2, and KIT. We did a phase I study to define the maximum tolerated dose and to assess the pharmacokinetics of SU6668 administered orally thrice daily with food. PATIENTS AND METHODS: Patients with histologically proven, advanced, and progressive solid tumors were included at a starting dose level of 400 mg/m2 thrice daily. The early onset of dose-limiting toxicities (DLT) required dose reductions to 100 and 200 mg/m2 thrice daily. Pharmacokinetics was done on days 1, 28, and 56. RESULTS: Sixteen patients were included. Two of the first three patients developed DLTs, which consisted of grade 4 fatigue and grade 3 serositis-like pains. Six patients at dose level 100 mg/m2 thrice daily experienced no DLT. At dose level 200 mg/m2 thrice daily, two out of seven patients experienced DLTs consisting of grade 3 abdominal pain, grade 4 anorexia and grade 3 nausea/vomiting. Increasing doses resulted in a disproportional increase in area under the curve and C(max) (peak plasma concentration). Both variables, however, decreased significantly on days 28 and 56 compared with day 1 (P < 0.05). No objective responses were observed. Acute phase response, probably mediated by interleukin-6, was observed in serial blood samples. CONCLUSIONS: The maximum tolerated dose of SU6668 given orally, thrice daily under fed conditions, is 100 mg/m2. Because of the low plasma levels reached at this dose level, the efficacy of SU6668 as a single agent is not to be expected.

Advanced glycation end products in human cancer tissues: detection of Nepsilon-(carboxymethyl)lysine and argpyrimidine.

Tumors are generally characterized by an increased glucose uptake and a high rate of glycolysis. Since one consequence of an elevated glycolysis is the nonenzymatic glycation of proteins, we studied the presence of advanced glycation end products (AGEs) in human cancer tissues. We detected the presence of the AGEs N(epsilon)-(carboxymethyl)lysine (CML) and argpyrimidine in several human tumors using specific antibodies. Because AGEs have been associated with the etiology of a variety of different diseases, these results suggest that CML and argpyrimidine could be implicated in the biology of human cancer.

Reduced growth, increased vascular area, and reduced response to cisplatin in CD13-overexpressing human ovarian cancer xenografts.

PURPOSE: Expression of aminopeptidase N/CD13 can be detected in several solid tumor types. Thus far, the role of CD13 in ovarian cancer has not been studied. We have investigated the expression pattern and biological function of CD13 in ovarian cancer. EXPERIMENTAL DESIGN: First, we studied the expression of CD13 in ovarian cancer tissue of 15 patients representing three different histological types (5 patients each) by immunohistochemistry. We then stably transfected the IGROV-1 human ovarian cancer cell line with a CD13 expression vector and examined the biological effect of CD13 in vitro and in vivo. RESULTS: The expression of CD13 in ovarian cancer was associated with the histological subtype: CD13 expression in tumor cells was observed in 80-100% of the patients with a serous or mucinous carcinoma and in only 20% of the clear cell carcinoma patients. In all patients' tumor samples, CD13-positive blood vessels were present. CD13 overexpression in IGROV-1 cells did not affect in vitro cell growth and sensitivity to doxorubicin, cisplatin, or gemcitabine. CD13 overexpression reduced invasion in Matrigel, which appeared to be independent of the aminopeptidase activity of CD13. Furthermore, the growth rate of IGROV-1/CD13 xenografts was reduced. The area of the vessel lumens was enlarged in a small percentage of vessels in the CD13-overexpressing xenografts. In addition, the CD13-overexpressing tumors were less sensitive to cisplatin. CONCLUSIONS: CD13 is expressed in tumor as well as endothelial cells in human ovarian cancer. Our results suggest that CD13 overexpression affects ovarian cancer growth, vascular architecture, and response to chemotherapy. Further elucidation of the mechanism of the observed effects of CD13 is warranted to better understand its role in the pathophysiology of ovarian cancer.

Soluble aminopeptidase N/CD13 in malignant and nonmalignant effusions and intratumoral fluid.

PURPOSE: On the basis of the finding of marked overexpression in angiogenic microvessels, aminopeptidase N/CD13 has recently been suggested to play a prominent role in tumor angiogenesis. A soluble form of CD13 (sCD13) is present in human plasma, but its role in cancer has not been addressed. We hypothesized that sCD13 would be shed by tumor cells and/or endothelial cells lining tumor vessels, giving high levels of sCD13 in intratumoral fluid (TF) deposits and in malignant effusions. If so, sCD13 could be a convenient potential marker for tumor load and/or activated tumor endothelium. EXPERIMENTAL DESIGN: We have measured the specific sCD13 activity in effusions from 90 cancer patients and 12 patients with a nonmalignant condition, and studied its relationship with other major (anti-)angiogenic factors. In a separate group of patients (n = 41), the relationship of sCD13 activity in plasma with tumor load was studied. RESULTS: The sCD13 activity was highest in plasma from cancer patients 71.9 (fmol/ml/s hydrolyzed substrate) versus 42.4 for healthy subjects. In TF, malignant effusions, and nonmalignant effusions, the activities were 52.8, 33.5, and 18.6, respectively. We further studied the relationship of sCD13 with tumor load as well as with vascular endothelial growth factor (VEGF), endostatin, matrix metalloproteinase (MMP)-2, MMP-9, urokinase-type plasminogen activator, and plasmin. A significant correlation of sCD13 activity in plasma was found with tumor load (r = 0.68; P = 0.01), suggesting that plasma sCD13 is, at least, partly originating from tumor(-endothelium). The concentrations of VEGF and endostatin and the activities of urokinase-type plasminogen activator and MMP-9, but not MMP-2, were significantly higher in TF compared with all other effusions. In TF, a correlation between sCD13 and VEGF was found (r = 0.67; P = 0.03). No correlation of sCD13 with the other protease activities was found. CONCLUSION: The sCD13 activity is elevated in plasma and effusions of cancer patients. A strong correlation of plasma sCD13 with tumor load was found. On the basis of these results, the potential of sCD13 activity as a tumor and/or angiogenesis marker warrants further investigation.

Efficacy and toxicity of the angiogenesis inhibitor SU5416 as a single agent in patients with advanced renal cell carcinoma, melanoma, and soft tissue sarcoma.

INTRODUCTION: Vascular endothelial growth factor (VEGF) is a key regulator in angiogenesis. Preclinical and clinical data support the role of VEGF and angiogenesis in renal cell carcinoma (RCC), melanoma (M), and soft tissue sarcoma (STS). The tyrosine kinase inhibitor SU5416 is a potent inhibitor of the VEGF receptors 1 and 2. EXPERIMENTAL DESIGN: We investigated 145 mg/m(2) SU5416 twice weekly in patients with advanced or metastatic RCC, M, and STS. The primary objectives were efficacy and safety. Disease assessments were performed after 4 and 8 weeks of treatment and every 2 months thereafter. Documented stable disease (SD) lasting for > or =3 months was considered an antitumor response. RESULTS: A group of 29 patients was entered in the RCC trial, 20 patients in the M trial, and 31 patients in the STS trial. Response was observed in 6 (1 minor response and 5 SDs) of 24 evaluable patients (25%) in the RCC group, 6 (1 minor response and 5 SDs) of 26 patients (23%) in the STS group, and none of the patients in the M group. Progression-free survival ranged from 7 to 252 days (median 59 days) in the RCC group, from 7 to 260 days (median 60 days) in the STS group, and from 14 to 139 days (median 41 days) in the M group. Toxicities observed were those reported previously for SU5416. CONCLUSION: SU5416 single agent is well tolerated. The antitumor response was low in patients with RCC and STS, whereas no responses were seen in patients with M.

Direct activation of caspases by RGD-peptides may increase drug sensitivity of tumour cells.

The realization that cellular homeostasis is dependent on the continuous integration of survival and death signals picked up from the environment and the recent advances in identifying the molecular players involved in these networks may increase our ability to manipulate apoptosis for therapeutic purposes. A recent paper by Buckley et al. in Nature(1)brings this goal one step closer by identifying peptides, containing the motif arginine-glycine-aspartate (RGD), that induce apoptosis by direct activation of caspase-3. We put this finding in the context of what is known about the RGD motif in the light of cancer treatment and suggest the possibility of a synergistic action with anticancer drugs. Copyright 1999 Harcourt Publishers Ltd.

Neurotoxic complications of chemotherapy in patients with cancer: clinical signs and optimal management.

Neurotoxic side effects of chemotherapy occur frequently and are often a reason to limit the dose of chemotherapy. Since bone marrow toxicity, as the major limiting factor in most chemotherapeutic regimens, can be overcome with growth factors or bone marrow transplantation, the use of higher doses of chemotherapy is possible, which increases the risk of neurotoxicity. Chemotherapy may cause both peripheral neurotoxicity, consisting mainly of a peripheral neuropathy, and central neurotoxicity, ranging from minor cognitive deficits to encephalopathy with dementia or even coma. In this article we describe the neurological adverse effects of the most commonly used chemotherapeutic agents. The vinca-alkaloids, cisplatin and the taxanes are amongst the most important drugs inducing peripheral neurotoxicity. These drugs are widely used for various malignancies such as ovarian and breast cancer, and haematological cancers. Chemotherapy-induced neuropathy is clearly related to cumulative dose or dose-intensities. Patients who already have neuropathic symptoms due to diabetes mellitus, hereditary neuropathies or earlier treatment with neurotoxic chemotherapy are thought to be more vulnerable for the development of chemotherapy-induced peripheral neuropathy. Methotrexate, cytarabine (cytosine arabinoside) and ifosfamide are primarily known for their central neurotoxic side effects. Central neurotoxicity ranges from acute toxicity such as aseptic meningitis, to delayed toxicities comprising cognitive deficits, hemiparesis, aphasia and progressive dementia. Risk factors are high doses, frequent administration and radiotherapy preceding methotrexate chemotherapy, which appears to be more neurotoxic than methotrexate as single modality. Data on management and neuroprotective agents are discussed. Management mainly consists of cumulative dose-reduction or lower dose-intensities, especially in patients who are at higher risk to develop neurotoxic side effects. None of the neuroprotective agents described in this article can be recommended for standard use in daily practise at this moment, and further studies are needed to confirm some of the beneficial effects described.

A doxorubicin-CNGRC-peptide conjugate with prodrug properties.

There is increasing interest in the exploitation of molecular addresses for the targeting of tumor imaging or therapeutic agents. A recent study demonstrated anticancer activity in human xenografts of doxorubicin (DOX)-peptide conjugates targeted to the tumor vascular endothelium, among them DOX coupled to the cyclic pentapeptide CNGRC [Science 279 (1998) 377]. In order to learn more about the mechanism of action of this type of DOX-peptide conjugates, we have studied the interaction of DOX-CNGRC with primary human umbilical cord vein endothelial cells (HUVEC) and tumor cells under defined in vitro conditions. We used a DOX conjugate, in which the cyclic CNGRC peptide, for which an in vivo endothelial address has recently been identified as aminopeptidase N (APN)/CD13, has been coupled via a hydrolysable spacer to the C-14 anthracycline-side chain. First we determined that the t(1/2) of DOX-CNGRC conjugate in human blood was 442 min (at 37 degrees ) allowing sufficient time for endothelial targeting when administered i.v. When cultured cells were exposed for 30 min to DOX-CNGRC a more cytoplasmic localization of fluorescent drug was seen when compared to DOX exposure and intracellular DOX-CNGRC was identified after extraction from the cells. This revealed differences in the cellular uptake process of the conjugate compared to DOX. The antiproliferative effect of DOX-CNGRC was determined by 30 min exposure in medium with a high protein content in order to mimick the in vivo targeting situation. In this medium, the IC(50) was 1.1 microM for highly CD13 expressing HT-1080, 1.45 microM for CD13 negative SK-UT-1 sarcoma cells and 6.5 microM for CD13 positive HUVEC. The IC(50) of DOX for these cells were 1.0, 2.0 and 7.3 microM, respectively. Although DOX-CNGRC inhibited the peptidase activity of CD13 up to 50%, our data do not favor an important role for the enzyme inhibition in the cytotoxic effect of the conjugate. The antitumor activity was tested in nude mice bearing human ovarian cancer xenografts (OVCAR-3). A weekly i.v. administration (3mg/kg DOX-equivalent, 3x) showed a minor (40%) growth delay, which does not indicate efficacy better than that expected for free DOX. In conclusion, this study indicates that the antiproliferative and anti-angiogenic effects of DOX-CNGRC as reported before, are likely caused by the cytostatic effects of intracellularly released parent drug DOX, independent of CD13 expression/activity. More research is needed to identify the optimal specific chemical configuration of DOX-peptide conjugates for in vivo targeting and receptor-mediated cellular uptake.

The effect of fluoropyrimidines with or without thymidine phosphorylase inhibitor on the expression of thymidine phosphorylase.

Thymidine phosphorylase (platelet-derived-endothelial-cell-growth-factor) catalyzes the reversible phosphorolysis of thymidine to thymine and 2-deoxyribose-1-phosphate, activates 5'-deoxy-5-fluorouridine (5'DFUR) and inactivates trifluorothymidine (TFT). The effect of 5'DFUR and TFT with or without a specific thymidine phosphorylase inhibitor (TPI) on thymidine phosphorylase mRNA, protein expression and activity was studied, in three human colon cancer cell lines, WiDR, HT29 and Lovo exposed for 72 h at IC50 concentrations. In Lovo cells TFT plus TPI only increased thymidine phosphorylase-protein expression 1.7-fold; 5'DFUR and TFT treatment increased thymidine phosphorylase mRNA levels 5- and 1.4-fold, respectively. In WiDR cells, 5'DFUR plus TPI significantly decreased thymidine phosphorylase-protein. TFT and TFT plus TPI increased thymidine phosphorylase-protein 2- and 3-fold, respectively. TPI and 5'DFUR decreased thymidine phosphorylase-mRNA levels significantly. In HT29 cells, 5'DFUR and 5'DFUR plus TPI decreased both thymidine phosphorylase-protein and thymidine phosphorylase-mRNA. In all cell lines 5'DFUR and TFT did not affect thymidine phosphorylase activity, but treatment with TPI (alone or in combination) eliminated thymidine phosphorylase activity. This demonstrated that regulation is drug and cell line dependent.

Glioblastoma-induced inhibition of Langerhans cell differentiation from CD34(+) precursors is mediated by IL-6 but unaffected by JAK2/STAT3 inhibition.

AIMS: Langerhans cell (LC) infiltration has been observed in glioblastoma, but the glioblastoma microenvironment may be conditioned to resist antitumor immune responses. As little is known about how glioblastoma may affect dendritic cell differentiation, here we set out to delineate the effects of glioblastoma-derived soluble factors on LC differentiation. METHODS: CD34(+) precursor cells of the human myeloid cell line MUTZ-3 were differentiated into LC in the presence of conditioned media of the human glioblastoma cell lines U251 or U373 and phenotypically and functionally characterized. RESULTS: Glioblastoma-conditioned media inhibited LC differentiation, resulting in functional impairment, as determined by allogeneic mixed leukocyte reactivity, and induction of STAT3 activation. IL-6 blockade completely abrogated these glioblastoma-induced immunosuppressive effects and reduced STAT3 phosphorylation. However, neither addition of JSI-124 (cucurbitacin-I; a JAK2/STAT3 inhibitor), nor of GW5074 (a Raf-1 inhibitor), both of which interfere with signaling pathways reported to act downstream of the IL-6 receptor, prevented the observed inhibitory effects on LC differentiation. CONCLUSION: Glioblastoma-derived IL-6 is responsible for the observed suppression of LC differentiation from CD34(+) precursors but appears to exert this effect in a STAT3 and Raf-1 independent fashion.

Phase I safety, pharmacokinetics, and inhibition of SRC activity study of saracatinib in patients with solid tumors.

PURPOSE: This dose-escalation study evaluated the safety, tolerability, and pharmacokinetics (PK) of the oral Src inhibitor saracatinib (AZD0530) in patients with advanced solid malignancies. Tumor biopsy samples were taken to investigate the effect of saracatinib on Src activity in tumors. EXPERIMENTAL DESIGN: Part A of the study followed a multiple-ascending dose design to establish the maximum tolerated dose (MTD) of saracatinib. Part B was a randomized, parallel-group, cohort-expansion phase to further assess tolerated doses. Safety, tolerability, and Src activity (immunohistochemistry and lysate-based methodologies) were assessed after 21 days of once-daily oral dosing. PK was assessed after single and multiple dosing. RESULTS: In part A, 30 patients received once-daily saracatinib at doses of 60 to 250 mg; the MTD was established as 175 mg. In part B, 51 patients were randomized to receive 50 mg (n = 16), 125 mg (n = 16), or 175 mg (n = 19) of saracatinib. The most common grade ≥3 events considered to be treatment related were anemia, diarrhea, and asthenia. Tumor Src activity was reduced following saracatinib treatment. The area under the concentration-time curve and C(max) of saracatinib increased with increasing dose. Saracatinib accumulated 4- to 5-fold on once-daily dosing to reach steady-state exposure after 10 to 17 days of dosing. The half-life was ∼40 hours. CONCLUSIONS: Saracatinib was well tolerated in patients with advanced solid malignancies. A reduction in tumor Src activity was observed. PK data show that saracatinib is suitable for once-daily oral dosing. Based on this study, the recommended dose for the phase II studies was chosen to be 175 mg/d.

Phase I evaluation of cediranib, a selective VEGFR signalling inhibitor, in combination with gefitinib in patients with advanced tumours.

AIM: Cediranib is a highly potent inhibitor of vascular endothelial growth factor receptor (VEGFR) signalling. Preclinical and clinical data suggest that inhibition of the VEGFR and epidermal growth factor receptor (EGFR) pathways may be synergistic. Combination treatment with cediranib and gefitinib, an EGFR signalling inhibitor, was evaluated in patients with advanced solid tumours. PATIENTS AND METHODS: Ninety patients received treatment in this four-part, open-label study (NCT00502060). The patients received once-daily oral doses of cediranib (20-45mg) and gefitinib 250mg (part A1; n=16) or 500mg (part B1; n=44). A cohort expansion phase investigated the potential pharmacokinetic interaction of cediranib 30mg with gefitinib 250mg (part A2; n=15) or 500mg (part B2; n=15). The primary objective was to assess the safety and tolerability of cediranib with gefitinib. Secondary assessments included pharmacokinetics, efficacy and pharmacodynamics. RESULTS: Combination treatment was generally well tolerated; the protocol-defined maximum-tolerated dose of cediranib was 30mg/day with gefitinib 250mg/day (part A1) and cediranib 45mg/day was the maximum dose investigated with gefitinib 500mg/day (part B1). The most common adverse events were diarrhoea (84 [93%]), anorexia (63 [70%]) and fatigue (60 [67%]). Cediranib pharmacokinetic parameters were not substantially different when given alone or in combination with gefitinib. Gefitinib pharmacokinetic parameters were similar to those seen previously with gefitinib monotherapy. Efficacy results included eight (9%) confirmed partial responses (6 renal; 1 lung; 1 osteosarcoma) and 38 (42%) patients with stable disease. Pharmacodynamic assessments demonstrated changes in levels of VEGF and soluble VEGFR-2 following treatment. CONCLUSIONS: Combination treatment was generally well tolerated and showed encouraging antitumour activity in patients with advanced solid tumours. These results merit further exploration.

Tyrosine kinase inhibitors of VEGF receptors: clinical issues and remaining questions.

The number of VEGFR tyrosine kinase inhibitors (TKIs) used as an anti-cancer agent is rapidly increasing, but several issues in clinical practice remain to be elucidated. VEGFR TKIs are multikinase inhibitors that have additional targets such platelet-derived growth factor receptors, which may result in an increased efficacy as well as an increased toxicity. Efficacy in several cancers has been shown, but acquired resistance also occurs during treatment with this new class of drugs. Tumor response evaluation can be a challenge, because VEGFR TKIs can cause extensive tumor necrosis without a marked decrease in tumor size. Therefore, new response criteria and functional imaging techniques are required. In this review we will also focus on the specific toxicities and their management: hypertension, proteinuria, cardiac toxicity, fatigue, hypothyroidism, voice changes, gastrointestinal toxicity, cutaneous reactions, wound healing, hemorrhage and thromboembolic events, hematological toxicity and cerebral toxicity. Furthermore we will discuss some issues regarding the pharmacology and dosing of these drugs. This review may provide important information to clinicians who prescribe VEGFR TKIs to their patients.

Proteomics of the TRAP-induced platelet releasate.

Upon stimulation, platelets release the soluble content of their cytoplasmic granules along with microparticles. This sub-proteome is of interest since many of its constituents are associated with coagulation, (tumor) angiogenesis, cell growth and adhesion. Previously, differential - antibody-based - serum analysis has yielded information on the proteins released from platelets upon stimulation. A promising alternative strategy is formed by identifying the proteins released by freshly isolated platelets from blood using proteomics. Here we report on the analysis of the thrombin receptor activating peptide (TRAP)-induced releasate from 3 different volunteers using high resolution, high mass accuracy hybrid LTQ-FT mass spectrometry in a GeLC-MS/MS workflow. We obtained an activated platelet releasate proteome comprising a total of 716 identified proteins with 225 proteins present in the releasate of 3/3 volunteers. This core dataset is characterized by gene ontology mining and signal peptide analysis. Meta-analysis of our dataset and two published datasets of platelet a-granules and microparticles reveals that 55% of our platelet releasate proteins can be annotated using these previous platelet subproteome data, of the remaining releasate proteome 5% overlaps with a published platelet secretome while 40% of our data consists of novel releasate proteins. This high-accuracy activated platelet releasate proteome represents the largest and most comprehensive analysis to date. This approach offers unique possibilities to analyse the role of platelet-secreted proteins in physiology and in diseases such as atherosclerosis and cancer.